Fluorescence and Multiphoton Imaging Resolve Unique Structural Forms of Sterol in Membranes of Living Cells

Abstract

Although cholesterol is an essential component of mammalian membranes, resolution of cholesterol organization in membranes and organelles (i.e. lysosomes) of living cells is hampered by the paucity of nondestructive, nonperturbing methods providing real time structural information. Advantage was taken of the fact that the emission maxima of a naturally occurring fluorescent sterol (dehydroergosterol) were resolvable into two structural forms, monomeric (356 and 375 nm) and crystalline (403 and 426 nm). Model membranes (sterol:phospholipid ratios in the physiological range, e.g. 0.5-1.0), subcellular membrane fractions (plasma membranes, lysosomal membranes, microsomes, and mitochondrial membranes), and lipid rafts/caveolae (plasma membrane cholesterol-rich microdomain purified by a nondetergent method) contained primarily monomeric sterol and only small quantities (i.e. 1-5%) of the crystalline form. In contrast, the majority of sterol in isolated lysosomes was crystalline. However, addition of sterol carrier protein-2 in vitro significantly reduced the proportion of crystalline dehydroergosterol in the isolated lysosomes. Multiphoton laser scanning microscopy (MPLSM) of living L-cell fibroblasts cultured with dehydroergosterol for the first time provided real time images showing the presence of monomeric sterol in plasma membranes, as well as other intracellular membrane structures of living cells. Furthermore, MPLSM confirmed that crystalline sterol colocalized in highest amounts with LysoTracker Green, a lysosomal marker dye. Although crystalline sterol was also detected in the cytoplasm, the extralysosomal crystalline sterol did not colocalize with BODIPY FL C 5 -ceramide, a Golgi marker, and crystals were not associated with the cell surface membrane. These noninvasive, nonperturbing methods demonstrated for the first time that multiple structural forms of sterol normally occurred within membranes, membrane microdomains (lipid rafts/ caveolae), and intracellular organelles of living cells, both in vitro and visualized in real time by MPLSM.Cholesterol is essential for optimal membrane transport, receptor-effector coupling, cell recognition, and other eukaryotic cellular processes (reviewed in Ref. 1). Increasing evidence indicates that plasma membrane cholesterol is organized into lateral and transbilayer cholesterol-rich microdomains (reviewed in Ref.2) such as lipid rafts and caveolae (reviewed in Refs. 2-6). These domains contain proteins involved in multiple cellular functions including signaling and cholesterol transport. For example, overexpression of caveolin-1 in mice stimulates high density lipoprotein uptake via plasma membrane caveolae and markedly increases plasma high density lipoprotein-cholesterol (8). In contrast, caveolin-1-deficient mice are lean and resistant to diet-induced obesity but show hypertriglyceridemia and exhibit vascular abnormalities (9 -11). There is a strong association of cholesterol abnormalities with cytotoxicity, sickle cell acanthacytosis, Niemann-Pick C disease, Alzhe...

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

“…The micro-crystalline DHE phagocytosed by the cells was largely found to be in lysosomes (70) but breakdown of the microcrystals occured over time (~2-3 days) such that monomeric DHE was distributed throughout the cell into the plasma membrane, lipid storage droplets, endoplasmic reticulum, and other organellar membranes. This was also observed in organelles isolated from cells incubated for several days on DHE from ethanolic stock solution (6,8,70,102,129). As a result of the increased localized concentration of micro-crystalline DHE and its enhanced excimeric fluorescence, difficulties arise in detecting low to moderate amounts of monomeric DHE (Fig.…”

“…(16,(30)(31)(32)(33)(34)(35)(36)(37)(38). Although there is considerable discussion regarding the solubility and orientation of these synthetic fluorescent sterol probes in membrane bilayers, especially 22-NBD-cholesterol and 25-NBD-cholesterol, at low concentrations some are thought to mimic the distribution and orientation of cholesterol (16,(35)(36)(37) (64,(69)(70)(71). However, synthetic DHE prepared by this established method, especially that obtained from commercial sources, may contain a significant sterol impurity, varying from batch to batch, and as high as 20-40% (43,70,71).…”

“…micro-crystals, lipoproteins, LUV, or cyclodextrin complexes) may affect the distribution of fluorescent sterol to lipid rafts/caveolae or non-rafts (see Section 6.3 and respectively (26,27,70,136). DHE was codistributed similarly as cholesterol into sterol-rich and -poor domains (26,27,70,82,102,135,136,172,182,196). Lipid raft/caveolae associated receptors such as the oxytocin receptor are sensitive to the structure of the sterol with which it interacts and both cholesterol and DHE, but not other sterols, are effective in reconstituting activity of this highly sterol-sensitive plasma membrane protein in cholesterol-depleted lipid rafts/caveolae (89-91).…”

“…6B), distinct and much less intense than microcrystalline DHE observed in lysosomes upon culturing cells with DHE microcrystals. MPLSM images of cells supplement with DHE as LUV revealed that DHE was nonrandomly distributed into sterol-rich and poor-domains within the plasma membrane of living L-cells (70,203,204). …”

“…Recently, real-time multiphoton imaging of the naturally-occurring fluorescent sterol DHE in the plasma membrane of living cells (70,82,203,204) was used to examine sterol distribution directly with high optical sectioning capability (206)(207)(208)(209). While subcellular distribution (plasma membrane, lysosome, lipid droplet, etc) of DHE was highly dependent on the method of delivery (microcrystals, LUV, MβCD), DHE at the plasma membrane was found to be distributed non-randomly into sterol-rich and sterol-poor regions in plasma membranes of living cells with the size range of sterol-rich clustering domains estimated to be from 200 nm (limit of optical microscopy) to 565 nm (70,71,82,203). Due to the diffraction limit of resolution, there is uncertainty about whether these regions, as visible under laser scanning microscopy, are contiguous lateral domains (70,71,203) or represent artificial enhancement of the intensity as a result of microvillar/filipodia extensions and/or folding (210,211) of the plasma membrane.…”